Inhibition of a Transcriptional Pause by RNA Anchoring to RNA Polymerase[unreadable] [unreadable] HK022 uses a novel, RNA-based antitermination mechanism to express many essential genes. A nascent transcript of a viral sequence called put binds to the EC that catalyzed its synthesis and remains associated with it as the transcript continues to elongate. Association with put RNA modifies the EC so that it resists termination at downstream terminators. No other protein factor is absolutely required, other ECs in the same cell are unaffected, and there is no obvious terminator specificity. Modification of the EC by put RNA also suppresses transcriptional pausing at a U-rich sequence located very close to the put site, and we have investigated the mechanism of put-mediated antipausing at this site. We showed that the U-rich sequence promotes "backtracking" of the EC. Backtracking is a retrograde movement during which the nucleotides at the 3'-end of the transcript are melted from the template DNA strand and extruded from RNAP, while compensating amounts of upstream DNA and nascent RNA reenter the EC. Backtracking increases the strength of certain pauses. In vitro transcription and footprinting assays revealed that put RNA suppresses pausing at the U-rich pause by limiting the extent of backtracking. Backtracking is limited because bound put RNA restricts re-entry of the nascent transcript into the RNA exit channel. The restriction is local and relaxes as the transcript elongates. The number of nucleotides over which the restriction operates allows us to use RNA as a "molecular tape measure" to locate the put binding site. We found that putL RNA binds to the surface of polymerase within 10 to 28 of the RNA exit channel, a region that includes amino acid residues known to be important for antitermination and RNA binding. Although binding is essential for antipausing and antitermination, these two activities of put differ: antipausing is limited to the immediate vicinity of the putL site, but antitermination is not. [unreadable] [unreadable] Transcriptional pausing is an important component of many regulatory networks and our knowledge about these networks is growing. For example, in a large number of Drosophila embryo genes, ECs are stalled near the transcription start site, possibly waiting for activation at later developmental stages. Backtracking is a mechanism that accounts for many known pauses, and RNA anchoring to the EC is potentially a general mechanism to regulate backtracking-associated pauses. Moreover, since the effect of anchoring is local, individual pauses can be targeted. Examination of the properties of several well-studied pause sites leads us to propose that RNA anchoring to RNAP is a widespread mechanism of pause regulation.[unreadable] [unreadable] Analysis of a bacteriophage parasitizing a commensal anaerobe [unreadable] [unreadable] We have sequenced and annotated the genome of bacteriophage B40-8, whose host is Bacteroides fragilis, a human commensal anaerobe that is frequently pathogenic. B40-8, a member of the Siphoviridae, has a circular double stranded DNA genome of 45,805 basepairs. The genome encodes 64 real or potential proteins, of which only 18 can be assigned functions based on homology, structural predictions or N-terminal sequencing of capsid proteins. The predicted open reading frames are oriented in the same direction and generally conform to the temporal cassette organization of terminase-head-tail-lysis, except that the major tail gene is found after the terminase and before the head genes. The distribution of numerous predicted promoters, terminators and conserved sequence motifs within the intergenic regions suggests a complex regulatory network of multiple overlapping transcripts similar to that of Streptomyces phage phiC31. A paucity of discernable Shine-Dalgarno sequences and the presence of a conserved element in their place suggest a different mechanism of translational initiation than that usually found in prokaryotes. The presence of submolar restriction fragments of the virus chromosome, alignment of other terminase large subunit coding sequences with CDS2 and other evidence strongly support a processive headful DNA-packaging mechanism. Sequencing of the chromosomal ends places the first cut at basepair 12013 and the second cut between base 17491 and 17753, which generates a first headful of approximately 51400 basepairs and 10.9% terminal redundancy. Thus B40-8 packaging is distinct from that of other well-studied phages, as the first cut site is not within but almost 10 kb downstream of the putative terminase genes. PCR analysis of purified phage demonstrate that host DNA is also packaged into capsids, and sequencing of genomic clones suggest that about 1% of the phage particles contain host DNA.[unreadable] [unreadable] [unreadable] Action of a sequence-specific transcript termination factor[unreadable] [unreadable] The Nun protein of phage HK022 protects lysogens against superinfection by phage lambda. It does so by binding to and terminating ECs that transcribe lambda DNA. Binding is promoted by nascent transcripts of the phage nutL and nutR sites, which are specifically recognized by Nun. In vitro, Nun arrests transcript elongation but does not by itself dissociate the arrested ECs. However, in vivo, Nun dissociates ECs that have transcribed the nutR site, presumably with the help of cellular termination factors, such as Mfd and/or Rho, that are known to act on arrested ECs. We have asked what the requirements are for Nun-assisted dissociation of the EC in vivo. To answer this question, we use Northern blots to measure the abundance and stability of Nun-terminated transcripts containing the nutL site and initiated at the lambda pL promoter. We reasoned that dissociation of the EC should allow cellular 3'-5' exonucleases, the major catalysts of mRNA degradation in E. coli, access to the 3' end of the terminated transcript. By contrast, simple arrest will lead to a transcript with a protected 3' end (because it is inside the EC), and this is likely to increase transcript stability. In addition, the arrested EC will sterically block transcription by following ECs, which should therefore back up on the template and eventually occlude the promoter ("constipation") . Therefore, factors that are required for dissociation of arrested ECs are likely to reduce the stability of Nun-terminated transcripts while increasing their rate of synthesis. We are currently determining the effects of inactivating Mfd, Rho, and cellular 3'-5' exonucleases on the abundance and stability of such transcripts. We also will determe the effect of changing the distance between the promoter and the nutL site since other work has suggested that dissociation is favored when the distance is increased. Our initial experiments show that in cells with the wild type pL-nutL distance (32 bp) Nun increases the half-life of PL mRNA more than 5-fold while decreasing its abundance about 2-fold. As expected, transcripts produced in the presence of Nun have 3' ends that are dispersed over about 100 nt downstream of nutL, while transcripts produced in its absence are considerably longer. This result argues that at least some transcripts are protected from degradation by undissociated, Nun-arrested ECs, and that constipation caused by Nun-mediated arrest reduces promoter activity. Inactivation of Mfd increases transcript stability about another 5-fold in the presence of Nun, but has no effect in its absence. Therefore Mfd does appear to promote dissociation of a fraction of the arrested ECs. We suggest that Mfd dissociates only those ECs that have lost the initiation factor sigma when arrest occurs. Current work suggests that sigma release occurs stochastically after an EC leaves the promoter, and it has been shown that sigma-containing ECs are resistant to the action of Mfd.